Compositions and methods for enhancing immune responses mediated by antigen-presenting cells

ABSTRACT

Molecular adjuvants are disclosed comprising an antigen presenting cell-targeting ligand functionally linked to an immunogen, e.g. tumor associated antigens, bacterial or viral antigens, etc. Methods are disclosed for delivery of these molecular adjuvants to patients, resulting in the transduction of activating signals to the targeted antigen presenting cell, thereby enhancing the immune response to the co-delivered immunogen.

This application is a divisional application of U.S. patent applicationSer. No. 09/051,685 filed Apr. 17, 1998, now U.S. Pat. No. 6,821,517which claims priority to PCT/US96/16825 filed Oct. 18, 1996, whichclaims priority to U.S. Provisional Patent Application No. 60/005,727filed Oct. 20, 1995. The foregoing applications are incorporated byreference herein.

The disclosure of commonly-owned, co-pending U.S. application Ser. No.08/299,285, issued Dec. 9, 1997 as U.S. Pat. No. 5,696,230, isincorporated by reference herein.

Pursuant to 35 U.S.C. §202(c), it is hereby acknowledged that the U.S.Government has certain rights in the invention described herein, whichwas made, in part, with funds from the National Institutes of Health,grant numbers CA 57362 and CA 36727.

FIELD OF THE INVENTION

The present invention relates to the field of vaccines and stimulationof acquired immunity. In particular, the present invention providesnovel compositions designed to deliver specific antigens to antigenpresenting cells and simultaneously deliver signals to those cells thatproduce a desired immune response.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by numerals inparenthesis in order to more fully describe the state of the art towhich this invention pertains. Full citations for these references arefound at the end of the specification. The disclosure of each of thesepublications is incorporated by reference herein.

The basis of acquired, specific immunity in an organism is the abilityto discriminate between self and non-self antigenic substances. Themammalian immune system uses cell surface molecules known as the majorhistocompatibility complex (MHC) for discriminating between self fromnon-self. There are two classes of MHC molecules: Class I molecules arefound on all nucleated cell types in the body; Class II molecules arefound mainly on cells involved in producing immune responses. Mostspecific immune responses are generated against peptides or peptidederivatives associated with MHC molecules.

The structure of MHC molecules is such that they naturally bind smallpeptides, glycopeptides, phosphopeptides, and the like. One importantfunction of MHC molecules is to bind peptides that are derived fromprocessed products of proteins expressed in cells expressing the MHCmolecules, and to transport these to the cell surface for display to theimmune system. In this way, some MHC molecules function to expose theimmune system to peptides that are representative of normal cellularproteins. This process occurs during development, when self is learned,and continues throughout the organism's lifespan. Different mechanismsof immune tolerance prevent the organism from responding to “self”peptides associated with MHC.

The introduction of non-self proteins into cells results in theappearance of new and different peptides in association with the MHCmolecules; these are recognized as “non-self,” resulting in an immuneresponse. For example, viral infection of a cell will result in theproduction of viral peptides expressed on the surface ofantigen-presenting cells in association with MHC molecules (generallyClass I MHC). Viral peptides presented with MHC molecules at the cellsurface will often be recognized as foreign and an immune response willbe mounted. Autoimmune disease can occur if tolerance to some selfpeptides is lost, or if immune response is produced against viral orother foreign proteins that cross react with normal peptides in the hostorganism.

In the case of bacterial infections or other insults from externalsources, new proteins or compounds enter the organism. Some cellsinvolved in the immune response are capable of phagocytosing foreignorganisms or proteins. These immune cells degrade (process) the proteinproducts and the derived peptides are expressed at the cell surface inassociation with MHC molecules, where a specific adaptive immuneresponse is generated against novel non-self components. This activityis called antigen processing and presentation and cells that mediatethis activity are called Antigen Presenting Cells (APC's). Manydifferent immune cell types, including macrophages, dendritic cells, Bcells, and other associated cell types, perform this function.

Antigen alone is often insufficient to produce an immune response.Sometimes, antigen must be presented with accompanying “signals” thatare mediated by ligand-receptor interactions between the APCs and theresponding lymphocytes or between these cells and soluble factors thatare present in the surrounding environment. The soluble factors includecytokines and other mediators of inflammation that are usually presentat sites of infections or insult (complement, kinins, other growth andcytokine factors). The signals can be positive in nature, resulting inlymphocyte proliferation and generation of an adaptive immune response,or negative in nature, resulting in apoptosis of responding lymphocytesand perhaps immune tolerance to that antigen. Antigen presentation oftenoccurs in the presence of helper T cells or other cell types thatsecrete arrays of cytokines, which influence or determine the type ofimmune response that is induced. At a cellular level, specific immuneresponses are generated in a mixed cellular environment that includesdifferent types of antigen presenting cells, helper T lymphocytes, othertypes of regulatory cells, and the responding lymphocytes (B cells forantibody responses and T cells for cellular responses). Directrecognition of peptides by T cells can also occur with some cell types,such as allografts, where the allogeneic MHC is directly recognized asforeign.

Antigen processing and its impact on types of immune responses tospecific antigens. The mechanism by which antigen is processed andpresented and the parameters that determine the types of immuneresponses that are generated (antibody versus cellular) are at presentnot well understood for many antigens. It is believed that there aredifferent classes of APCs that can produce different types of immuneresponses. In general, APC-induced responses to exogenous antigens thatare taken up by endocytosis are believed to be presented to the immunesystem in the context of Class II MHC and lead to recruitment of Thelper cells that interact with B cells and ultimately produce anantibody response. In contrast, endogenous peptides from cells associatewith MHC Class I molecules and produce cellular activities that includecytotoxic T lymphocytes (CTL) and Delayed Type Hypersensitivity (DTH)T-cells. There are important exceptions to these mechanisms. Forexample, many CTLs reactive with exogenous peptides have been described,and it is possible to generate CTLs to specific peptides that have beenadded to in vitro cultures of immune cells.

Other factors can determine the types of immune responses that aregenerated. For example, the nature of peptide association with MHC(either Class I or Class II) is an important factor that influencestypes of immune responses. In the case of Class I MHC molecules, thereare specific binding motifs for peptide association (Rammensee et al,Ann. Rev. Imm. 11: 213, 1993). Binding motifs have been established forH-2 K^(d), K^(k), D^(d), and other murine and human MHC. There are alsoparameters of peptide sequence that determine affinity for class II MHC.Thus, the types of peptides to which an individual can mount an immunesystem response are in part determined by the immunogenetic genotype andphenotype, which establish the shape and structure of the MHC moleculesexpressed by that individual.

In summary, the types of immune response that are generated in anorganism in response to antigenic challenge is the result of a myriad ofcontributing factors, including: the immunogenetic background of theindividual, prior sensitization to antigens, the route and form ofantigen exposure, age and gender of the organism, and other factors.Almost all acquired immune responses that involve specific T-cellrecognition are directed toward small peptides bound to the peptidebinding groove of MHC molecules, the obvious exception being theresponse to superantigens. Cellular immune reaction (T-helper reaction,CTL, DTH) to peptides bound to MHC are usually generated throughpresentation of the antigen to T cells by antigen-presenting cells(APCs).

Tumor Vaccines. Cancer cells express aberrant molecules known astumor-associated antigens. The immune system has the potential torecognize such structures as “foreign” and to mount specific immuneresponses against them, so as to reject tumor cells in much the same waythat an allograft is rejected. This provides the basis for interest inthe development of active specific immunotherapeutic (ASI) agents(cancer “vaccines”) based on cancer-associated antigens.

Early studies on rodent tumors induced by chemical carcinogens,ultraviolet radiation, or viruses showed induction of immunologicalrejection of secondary tumor challenge. Subsequent studies onspontaneous tumors showed that these animals were incapable of inducingimmune-mediated rejection of the tumor. Although a large number of humantumor-associated antigens have been characterized, most of these arealso expressed by some normal cells. Therefore, immunological toleranceto such molecules makes it difficult to stimulate responses against suchantigens. Moreover, it is a concern that induction of strong immuneresponses against self molecules may result in the development ofautoimmune disorders. Since tumor-specific antigens are seldom detectedin spontaneous cancers, approaches to develop active specificimmunotherapy for common cancers, based on tumor-associated antigens,have been viewed with pessimism.

Nonetheless, interest in tumor immunology and the development of ASI inparticular has persisted. There are at least four reasons for thecurrent interest in ASI approaches. First, cell-mediated immuneresponses have been recognized as the key factor in immunologicalrejection of cancer. T cells recognize processed peptides in associationwith major histocompatibility complex (MHC) molecules, so intracellularproteins can give rise to peptide targets for cell-mediated responses.Further, since antigen processing and presentation are critical steps inT cell recognition, cancer-associated alterations (in itspost-translational processing or levels of expression) of a self proteinmay result in presentation of novel peptide fragments on cancer cells.Secondly, tumor specific point mutations in certain genes have beencharacterized in several animal and human cancers. Some of thesemutations generate novel peptide fragments that bind MHC moleculesresulting in the production of new epitopes for recognition by T cells.This process allows for the induction of specific immune responsesagainst cancer cells carrying such mutations. Third, manipulation ofimmune responses using cytokines, mutated antigens, and other means havesometimes resulted in tumor rejection even in cases of tumors thatexpress weakly immunogenic antigens. Fourth, some individuals withsevere immunodeficiencies have a higher incidence of tumors than thenormal population, suggesting that the immune system plays an importantrole in eliminating some tumors.

Various methods have been utilized for stimulating general immuneresponses, especially for non-antigenic or weakly antigenic substancesof interest. For example, adjuvants, such as complete Freund's andRibi's, have long been used for this purpose. These adjuvants compriseoily solutions containing components, such as lipopolysaccharides thatstimulate generalized immune responses. It is believed that the oilssurround a water-soluble antigen, such as a peptide, thereby protectingit from degradation in the body and facilitating phagocytosis andpassage through cell membranes of antigen presenting cells.

Another approach to stimulating the immunogenicity of a weakly-antigenicpeptide or protein has been to couple the weak antigen to a carrierprotein that is known to be a good immunogen. Common carrier proteinsinclude keyhole limpet hemocyanin, fowl gamma-globulin and bovine serumalbumin. Alternatively, the immunogencity of a weak antigen may beenhanced by polymerizing it into large aggregates by way ofcross-linking agents, such as glutaraldehyde. Both these methods rest onthe notion that a weak antigen coupled to a strong antigen will enhancethe generalized immune response. In a similar method, solid-phase resinsand peptide synthetic methods may be employed to synthesize a peptiderepeatedly, to form a highly-branched complex. Again, the basis for thisapproach is to present the antigen in very unusual (and very “non-self”)context to the immune system, to stimulate antibody production.

In yet another approach, a weakly antigenic protein or peptide isattached to a solid particle such as a latex bead or resin. The purposeof this approach is to enhance phagocytosis of the antigen bymacrophages. Additionally, peptides and proteins have been encapsulatedin liposomes to enhance passage through membranes of antigen presentingcells, to enhance phagocytosis and to stimulate generalized immuneresponses because of the “non-self” characteristics of the liposomecarrier.

The approaches described above have met with varying degrees of successin stimulating the immunogenicity of weakly antigenic or non-antigenicsubstances. However, they provide only a generalized stimulation ofimmunity, and are not designed to target specific populations of immunesystem cells (such as antigen presenting cells). A desired objective ineffecting therapeutic intervention in various disease states is toprovide a means for specifically targeting a protein or peptide to apopulation of antigen-presenting cells and thereby stimulate those cellsto internalize the antigen of interest and present it to the immunesystem in an effective, specific context. Insofar as it is known, such asystem is not currently available.

SUMMARY OF THE INVENTION

The present invention provides novel compositions and methods fordelivering specific antigens to antigen-presenting cells, andsimultaneously delivering signals to those cells that produce a desiredimmune response. The compositions of the invention are sometimesreferred to herein as “APC-targeted activating antigens.”

According to one aspect of the invention, these APC-targeted activatingantigens, which elicit an immune response mediated by anantigen-presenting cell, comprise at least one antigenic moietyfunctionally linked to at least one targeting moiety that bindsspecifically to a characteristic determinant on the antigen-presentingcell. For purposes of the present invention, the term “functionallylinked” is defined generally as linking of the moieties in such a waythat each moiety retains its intended function. This is particularlyrelevant with respect to the targeting moiety, which is designed to bindto a characteristic determinant on the antigen-presenting cell.

Antigen-presenting cells contemplated for targeting according to thepresent invention include, but are not limited to, monocytes, dendriticcells, macrophages, B cells and some T cells. In preferred embodimentsof the invention, the characteristic determinant on the selected APC isa cell surface receptor and the targeting moiety of the APC-targetedantigen is a ligand that binds to the receptor. It is particularlypreferred that the cell surface receptor be an immunomodulatoryreceptor. Suitable cell surface receptors include, but are not limitedto, C5a receptor, IFNγ receptor, CD21(C3d receptor), CD64 (FcγRIreceptor), and CD23 (FcεRII receptor).

One exemplary APC-targeted antigen of the invention is designed to bindto the C5a receptor, and the targeting moiety is a C5a receptor ligand,which is preferably a peptide analog of C5a corresponding to theC-terminal 10 residues of C5a. Another exemplary composition of thepresent invention is designed to bind to the IFNγ receptor, andcomprises a targeting moiety which is a IFNγ receptor ligand, preferablya peptide analog of IFNγ corresponding to the N-terminal 39 residues ofIFNγ.

The antigenic moiety of the APC-targeted antigens of the invention cancomprise essentially any antigenic substance, including, but not limitedto, peptides and proteins, glycopeptides and glycoproteins,phosphopeptides and phosphoproteins, lipopeptides and lipoproteins,carbohydrates, nucleic acids and lipids. The APC-targeted antigens cancomprise more than one antigenic moiety, and likewise can comprise morethan one targeting moiety. Moreover, these moieties can be functionallylinked in several fashions. For instance, if “T” represents a targetingmoiety, and “Ag” represents an antigenic moiety, the APC-targetedantigens of the present invention may be oriented as follows:

-   -   Ag-T;    -   T-Ag;    -   T₁-Ag-T₂;    -   T₁-[Ag]_(n)-T₂ (wherein [Ag]_(n) represents a multiplicity of        antigens.

Examples of the general formulas set forth above include:Ag-C5a agonist peptide;IFNγ peptide-Ag;IFNγ peptide-[Ag]_(n)-C5a agonist peptide.

According to other aspects of the present invention, methods areprovided for using the APC-targeted antigens of the invention. Theseinclude methods of activating an antigen-presenting cell with atargeting ligand and methods of eliciting an antigen presentingcell-mediated immune response in a subject in which such a response isdesired. General methods of immunizing or vaccinating a patientrequiring such treatment, methods of treating a tumor, and methods forproducing antibodies specific for a pre-determined antigen for use asresearch tools or for diagnostic purposes are also contemplated to bewithin the scope of the present invention.

The numerous features and advantages of the compositions and methods ofthe present invention are described more fully in the detaileddescription set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the antibody titer produced in miceimmunized with the indicated peptide constructs, as determined byradioimmunoassay, and shows the relationship between the amount of¹²⁵I-goat anti-mouse antibody bound vs. the dilution factor of mousesera which had been incubated in microtiter wells coated with the MUC1epitope peptide.

FIG. 2 is a graph illustrating the increase in antibody titer in thesera of mice collected either before (pre) or after immunization withpeptides 3 (YKQGGFLGLYSFKPMPLaR) (SEQ ID NO:2) and 4(YSFKPMPLaRKQGGFLGL) (SEQ ID NO:5) as determined by radioimmunoassay andshows the relationship between the amount of ¹²⁵I-goat anti-mouseantibody bound and the dilution factor of mouse sera which had beenincubated in microtiter wells coated with MUC1 epitope peptide. Notethat peptides 3 and 4 comprise two moieties, a targeting ligand and anantigen to which an immune response is desired.

FIG. 3 is a graph illustrating the titers of antibody classes andsubclasses produced in mice following immunization with peptide 3(YKQGGFLGLYSFKPMPLaR) (SEQ ID NO:2) as determined by ELISA using rabbitanti-mouse IgA, IgG1, IgG2a, IgG2b, IgG3, and IgM, followed by goatanti-rabbit conjugated to peroxidase and detected using p-nitrophenylphosphate cleavage monitored at 405 nm.

FIG. 4 is a graph illustrating the specificity of binding of theantibody subclasses in sera from mice immunized with peptide 3(YKQGGFLGLYSFKPMPLaR) (SEQ ID NO:2) as determined by ELISA using bindingto microtiter wells coated with MUC1 epitope peptide and detection withrabbit anti-mouse IgG2a, IgG2b, or IgM followed by incubation with goatanti-rabbit conjugated to peroxidase and detected using p-nitrophenylphosphate cleavage monitored at 405 nm.

DETAILED DESCRIPTION OF THE INVENTION

A major obstacle in the development of vaccines and otherimmunostimulatory agents is the inability of some antigens to be readilytaken up and processed by antigen presenting cells. Uptake of antigensby APCs is an essential step for stimulating an effective immuneresponse, since the immune system recognizes the antigen only after ithas been processed by the APC and presented on the surface of the APC inconjunction with the major histocompatibility complex (MHC).

It is known that APCs, including dendritic cells, monocytes, macrophagesand B cells, possess functional receptors for numerous molecules thatmodulate the immune response. It has now been discovered in accordancewith the present invention that ligands which bind to these receptorscan be conjugated to weakly immunogenic antigens for example, as a wayof delivering antigens to the antigen presenting pathway of the APC andsimultaneously activating the antigen presenting capacity of the APC.Thus, these conjugates bind to a receptor on the APC surface, transducea biological signal, and are internalized by the APC. The antigenicmoiety of the conjugate is thereby delivered to the antigen presentingpathway of the APC along with the simultaneous activation of the APC.

The above-described conjugates are sometimes referred to herein as“molecular adjuvants” or “APC-targeted activating antigens.” TheAPC-targeted activating antigens of the invention are designed to elicitimmune responses mediated by one or more types of antigen presentingcells. Accordingly, an APC-targeted activating antigen comprises atleast one antigenic moiety linked to a targeting and activating moietythat binds specifically to at least one characteristic determinant onthe selected antigen presenting cell type. This binding is followed byinternalization of the APC-targeted antigen and results in presentationof the antigen moiety on the surface of the APC. For purposes of thepresent invention, the term “antigenic moiety” may refer to anysubstance to which it is desired that an immune response be produced.The selected antigenic moiety may or may not be capable of eliciting animmune response by conventional means.

The term “determinant” is used herein in its broad sense to denote anelement that identifies or determines the nature of something. When usedin reference to an antigen presenting cell, “determinant” means thatsite on the antigen presenting cell which is involved in specificbinding by the targeting ligand moiety of the molecular adjuvant of theinvention.

The expression “characteristic determinant” as used herein, signifies anepitope (or group of epitopes) that serves to identify a particularpopulation of antigen presenting cells and distinguish it from otherantigen presenting cell populations. Cell-associated determinantsinclude, for example, components of the cell membrane, such asmembrane-bound proteins or glycoproteins, including cell surfaceantigens, histo-compatibility antigens or membrane receptors.

The expression “specific binding”, as used herein refers to theinteraction between the targeting ligand moiety and a characteristicdeterminant on the antigen presenting cell population sought to beactivated in accordance with this invention, to the substantialexclusion of determinants present on other cells.

Certain exemplary compositions of the invention have been synthesized,and have been shown to elicit APC-mediated immune responses inaccordance with the mechanisms described above. For example, antigenicepitopes have been conjugated to the amino-terminal end of a C5adecapeptide agonist capable of binding to C5a receptors present on thesurface of many APCs. Mice that were inoculated with an epitope of humanMUC1 (a cell surface-associated mucin) conjugated to such a C5a agonistexhibited pronounced antibody titers against the MUC1 epitope, includinghigh titers of specific antibodies with isotypes IgG2a and IgG2b. Micethat were inoculated with (1) MUC1 epitope alone, (2) C5a agonist alone,(3) unconjugated MUC1 epitope and C5a agonist together, or (4) C5aagonist conjugated to MUC1 epitope in a manner in which the biologicalactivity of the C5a moiety was blocked, did not express a significantspecific immune response. These results are described in greater detailin Example 1. Similar results were observed with conjugates of C5aagonist to a 12 kDa polypeptide, serum amyloid A (SAA), as described ingreater detail in Example 2. These data tend to demonstrate thefeasibility of the invention, which is to use receptor-binding ligandsas a way to deliver antigens to APCs, with the simultaneous activationof APCs by the ligand moiety.

As described in greater detail below, the C5a receptor is only one ofmany receptors expressed on APCs. This invention encompasses the use ofvarious ligands with selectivity to other receptors that mediate signaltransduction events in the APCs, to be used alone or in conjunction withC5a agonists to influence the nature of immune response generated, i.e.,humoral, cellular, Th1, Th2, and the like. Vaccines and otherimmunotherapeutic agents can be prepared with an array of such targetingmoieties that serve to target the antigen moiety to a specificpopulation of APCs and simultaneously activate these and other cellsinvolved in various immune modulatory pathways.

The detailed description below sets forth preferred embodiments formaking and using the targeted antigens of the present invention. To theextent that specific compounds and reagents are mentioned, these are forthe purposes of illustration, and are not intended to limit theinvention. Any biochemical, molecular or recombinant DNA techniques notspecifically described are carried out by standard methods, as generallyset forth for example, in Ausubel et al., “Current Protocols inMolecular Biology,” John Wiley & Sons, Inc., 1995.

I. Preparing APC-Targeted Activating Antigens

A. Selection of Components

Antigen presenting cells have various receptors on their surfaces forknown ligands. Binding of ligands to these receptors results in signaltransduction events that stimulate immune or tolerance responses. Manyof these receptors are known to internalize and recycle in the cell.Others are suspected of doing the same. As such, these receptors areideal targets for delivering antigens and activation signalssimultaneously to APCs.

As discussed above, APCs include several cell types includingmacrophages, monocytes, dendritic cells, B cells, some T cells and otherpoorly characterized cell types. It is believed that these differentclasses of APCs can produce different types of immune responses.Accordingly, by targeting a receptor prevalent on a specific populationof APCs, a particular desired immune response may be favored. Anexemplary list of receptors contemplated for targeting in the presentinvention, and the rationale for their selection, is set forth below.These APC receptors are particularly appropriate for use in the presentinvention based on the following criteria: they are receptors expressedon APCs; the receptors are internalized upon binding of ligand; thereceptors can transmit signals in the cells that influence antigenprocessing and presentation by these cells; some of the receptors arebelieved to be involved in signaling Th1 type cellular responses,whereas others are predicted to generate Th2 type humoral responses. Thelist set forth below is not exhaustive, but merely representative of thetype of targeted receptors preferred in practicing the presentinvention. Other receptors, or other cell-surface characteristicdeterminants on antigen presenting cells may also be used as targets forthe targeted antigens of the present invention. The receptor or othercharacteristic determinant need not be directly involved in the immuneresponse.

C5a receptor. This receptor is preferred for use according to thepresent invention. It is present on PMNS, macrophages, dendritic cells,smooth muscle cells and some mast cells. A number of biologicalactivities have been ascribed to C5a, mostly associated withinflammatory and immune responses. According to a preferred embodiment,this invention relies on the capability of C5a, as a targeting ligand,to specifically bind to its cognate receptor, so as to activate antigenpresenting cells, including macrophages, monocytes and dendritic cells,through a G protein-mediated signal transduction pathway. Subsequent tosignal transduction, the receptor/ligand complex is internalized. In thecase of dendritic cells, C5a has been shown to induce a Th1 typeresponse.

IFNγ receptor. The interferon γ receptor is expressed on macrophages,monocytes, dendritic cells, other APCs, some B cells, fibroblasts,epithelial cells, endothelium, and colon carcinoma cells. IFNγ bindingto its receptor induces macrophage and dendritic cell activation, B celldifferentiation, and expression of MHC class I and class II molecules inmany cell types. The receptor is involved in signal transductionpathways. IFNγ is mainly produced in the body by activated T cells,particularly during the generation of Th1 type response. It is alsoproduced by CD8+ cytotoxic T lymphocytes following recognition ofantigen associated with MHC class I and by natural killer cellsstimulated with TNFα and microbial products (Barclay et al. 1993,).

CD 21 (C3d receptor). CD 21 is the receptor for the C3d complementfragment. It is a receptor for the Epstein-Barr virus and may be animportant interferon a receptor (Barclay et al., supra). CD 21 isexpressed on B cells, follicular dendritic cells, other APCs, pharyngealand cervical epithelial cells, and some thymocytes. It is involved inactivation and proliferation of B cells through a signal transductionmechanism and it has been associated with increases in antigenpresentation activities by those cells.

CD 64 (FcγRI receptor). CD 64 is a high affinity receptor for IgG, theonly known receptor that binds monomeric IgG (Barclay at al, supra).This receptor is found on macrophages, monocytes and other immune cellpopulations treated with IFNγ. The IgG₁, binding site resides in the CH2domain. IFNγ strongly upregulates expression of this receptor, which isthe primary receptor involved in antibody-dependent cell mediatedcytotoxicity reaction, and phagocytic activity by these cells.

CD 23 (FcεRII receptor). CD 23 is a low affinity receptor for IgE (notrelated to the high affinity IgE receptor found on basophils and mastcells). It is found on some B cell populations, macrophages,eosinophils, platelets, and dendritic cells (Barclay et al, supra). CD23 mediates IgE dependent cell mediated cytotoxicity and phagocytosis bymacrophages and eosinophils, and binding of IgE immunocomplexesincreases the efficiency of antigen processing and presentation by someAPCs, through a signal transduction mechanism that includes the p59 fyntyrosine kinase. The ligand for CD 23 is the Cε3 domain of IgE.

As mentioned above, the APC-targeted antigens of the present inventioncomprise at least one antigenic moiety and at least one targetingmoiety. The targeting moiety can be derived from naturally-occurringligands for a selected receptor on an APC, or analogs and derivatives ofsuch ligands. For instance, the C5a receptor is a preferred receptor foruse in practicing the present invention. Naturally-occurring C5a can beutilized as the targeting moiety in the APC targeted activating antigensof the invention. However, native C5a is not preferred for use as thetargeting moiety as it induces a myriad of pro-inflammatory responseswhich may have undesirable side effects. In particularly preferredembodiments of the invention, C-terminal C5a agonist analogs capable ofC5a receptor binding and signal transduction in a response selectivemanner are utilized. Such analogs are described in detail incommonly-owned U.S. application Ser. No. 08/299,285, the entiredisclosure of which is incorporated by reference herein.

An exemplary C5a C-terminal decapeptide agonist preferred for use in thepresent invention is:

YSFKPMPLaR (SEQ ID NO:1)This decapeptide is a potent agonist of naturally occurring C5a, and ispreferred to naturally occurring C5a because its small size contributesto ease of synthesis and solubility. Moreover, these conformationallybiased peptides are stable toward serum carboxypeptidase digestion,express a level biological selectivity, and have been shown to benon-toxic in high concentrations in athymic mice.

Peptide analogs of naturally-occurring interferon γ are alsocontemplated for use in the present invention. Peptides corresponding tothe amino terminal 39 amino acids of IFNγ have been shown to compete forbinding with native IFNγ. Antibodies against this domain blockbiological activity, and removal of the first 10 amino terminal residueseliminates biological activity. This suggests that binding of IFNγ toits cognate receptor is mediated by this portion of the molecule.Accordingly, peptides based on this domain are contemplated to be of usefor delivering antigens to APCs expressing IFNγ receptors. In thisregard, it should be noted that human and mouse IFNγ are absolutelyspecies specific in binding and activity. Consequently, for stimulatingAPC-mediated immune responses in mice, the mouse peptides will beutilized, and the human peptide will likewise be utilized forstimulating APC-mediated immune responses in humans. The mouse IFNγ 39amino acid peptide analog is composed of the following sequence:HGTVIESLESLNNYFNFFGIDVEEKSLFLDIWRNWQKDG (SEQ ID NO:3) The human IFNγ 39amino acid peptide analog is composed of the following sequence:

(SEQ ID NO:4) QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEE

Another ligand contemplated for use in the present invention is the C3dGcomponent of complement. This component is a 348 residue fragmentderived by proteolytic cleavage from the C3b precursor (residue 955-1303of C3; Swissprot accession p01024). C3dG can be converted to C3d(residues 1002-1303) and C3g (residues 955-1001). C3dG and C3d remainassociated with non-activator surfaces and serve as opsonins forphagocytosis by macrophages and other antigen presenting cells. Cd 21 isthe C3dG and C3d receptor.

The above-listed ligands exemplify the type of ligand preferred forpractice of the present invention. However, it will be appreciated bythose skilled in the art that other ligands may be utilized as thetargeting moiety of the APC-targeted antigens of the invention. Theseinclude ligands that are already known in the art, as well as ligandsthat may be discovered and developed henceforth. Antibodies or antibodyfragments also may be used to target APC-specific cell surface antigens.

The type of antigen that can be chosen as the antigenic moiety in thepresent invention can be any peptide, polypeptide or derivative thereoffor which an immune response or antibody production is desired. Theseinclude but are not limited to, peptides, polypeptides (i.e. proteins)and derivatives thereof, such as glycopeptides, phosphopeptides and thelike. Synthetic peptide and polypeptide derivatives or analogs, or anyother similar compound that can be conjugated to a receptor-targetingmoiety can be used in the present invention. Moreover, these peptides,proteins and derivatives may comprise single epitopes or multipleepitopes for generating different types of immune responses. Indeed, ifan entire protein is conjugated to a targeting moiety, this protein islikely to comprise numerous epitopes, which may vary depending upon thesolvent conditions and their effect on secondary and tertiarystructure-of the protein. Carbohydrates, nucleic acids and othernon-protein substances also may be used as the antigenic moiety. Methodsare available in the art for conjugating these substances to the peptideor protein targeting moiety.

In preferred embodiments of the invention, the antigenic moietycomprises agents that are weakly antigenic or non-antigenic undercurrently available immunization conditions. Many tumor-associatedantigens fall into this category, because the antigens also areexpressed by normal cells. Therefore, immunological tolerance to suchmolecules makes it difficult to stimulate responses against suchantigens. Other proteins that fall into this category include naturallyoccurring proteins from one species (e.g., human) for which it would bedesirable to produce antibodies in another species but which arerecalcitrant to antibody generation in the other species.

One well-characterized tumor antigen is a cell surface-associated mucinthat is highly overexpressed and differentially glycosylated bydifferent adenocarcinomas, including breast, pancreas, lung and prostatecarcinomas. Aberrant glycosylation of MUC1 by adenocarcinomas results inthe addition of some blood group carbohydrate antigens to this coreprotein and the exposure of epitopes which have been detected bymonoclonal antibodies on the core protein that are not exposed on formsof this protein produced by normal epithelial cells. A full-length cDNAsequence of human mucin-1 (MUC1) revealed an encoded protein with anaverage length of approximately 1200 amino acids (depending on thelength of the tandem repeat allele) with several obvious domains: anamino terminal signal peptide; a large domain made up of multipleidentical 20 amino acid tandem repeats flanked by several repeats thatcontain degenerate sequences; a hydrophobic-spanning domain of 31 aminoacids; and a cytoplasmic domain of 69 amino acids at the carboxylterminus. The most well-characterized tumor associated epitopesdescribed to date for MUC1 are found in the tandem repeat domain. Theseinclude carbohydrate structures and protein structures. MUC1 tumorassociated epitopes are well characterized, and thus have been proposedto be used for the production of tumor vaccines using conventionalmethods. Exemplary compositions of the present invention comprise MUC1epitopes, such as those set forth below, as the antigenic moiety of theAPC-1 targeted antigens of the invention, to demonstrate the potentialof the present invention as potent tumor vaccines.

MUC1 epitope predicted to bind to class I molecules of the H-2 k^(b)type has sequence homology to the juxtamembrane region of MUC1;

YKQGGFLGL (SEQ ID NO:6)

MUC1 tandem repeat has the sequence:

GVTSAPDTRRAPGSTAPPAH (SEQ ID NO:7)

The components comprising the APC-targeted antigens of the invention canbe made separately, then conjugated. For example, a small peptideanalog, such as the above-described C5a agonists, may be produced bypeptide synthetic methods, and conjugated to a protein which has beenpurified from naturally occurring biological sources. Alternativelyproteins engineered for expression via recombinant methods may be used.Additionally, targeted antigens comprising peptide components (i.e., apeptide antigenic epitope conjugated to a peptide receptor ligand) canbe synthesized in tandem by peptide synthetic chemistry according toknown methods and as described in greater detail below. Finally,targeted antigens of the invention comprising two larger polypeptidemoieties (i.e., a large polypeptide antigen linked to a large ligand)can be made by recombinant techniques. For example, DNA moleculesencoding both components can be ligated together by recombinant means,then expressed as the conjugated fusion protein. Methods of making thesedifferent types of compositions are set forth in greater detail below.

B. Peptides

Oligopeptides required for the present invention may be prepared byvarious synthetic methods of peptide synthesis via condensation of oneor more amino acid residues, in accordance with conventional peptidesynthesis methods. Preferably, peptides are synthesized according tostandard solid-phase methodologies, such as may be performed on anApplied Biosystems Model 430A peptide synthesizer (Applied Biosystems,Foster City, Calif.), according to manufacturer's instructions. Othermethods of synthesizing peptides or peptidomimetics, either by solidphase methodologies or in liquid phase, are well known to those skilledin the art. When solid-phase synthesis is utilized, the C-terminal aminoacid is linked to an insoluble resin support that can produce adetachable bond by reacting with a carboxyl group in a C-terminal aminoacid. One preferred insoluble resin support isp-hydroxymethylphenoxymethyl polystyrene (HMP) resin. Other usefulresins include, but are not limited to: phenylacetamidomethyl (PAM)resins for synthesis of some N-methyl-containing peptides (this resin isused with the Boc method of solid phase synthesis; and MBHA(p-methylbenzhydrylamine) resins for producing peptides havingC-terminal amide groups.

During the course of peptide synthesis, branched chain amino andcarboxyl groups may be protected/deprotected as needed, usingcommonly-known protecting groups. In a preferred embodiment, N^(α)-aminogroups are protected with the base-labile 9-fluorenylmethyloxycarbonyl(Fmoc) group or t-butyloxycarbonyl (Boc groups). Side-chain functionalgroups consistent with Fmoc synthesis may be protected with theindicated protecting groups as follows:

arginine (2,2,5,7,8-pentamethylchroman-6-sulfonyl); asparagine(O-t-butyl ester); cysteine glutamine and histidine (trityl); lysine(t-butyloxycarbonyl); serine and tyrosine (t-butyl). Modificationutilizing alternative protecting groups for peptides and peptidederivatives will be apparent to those of skill in the art.

C. Proteins

Full-length proteins for use in the present invention may be prepared ina variety of ways, according to known methods. Proteins may be purifiedfrom appropriate sources, e.g., human or animal cultured cells ortissues, by various methods such as gel filtration, ion exchangechromatography, reverse-phase HPLC and immunoaffinity purification,among others. However, due to the often limited amount of a proteinpresent in a sample at any given time, conventional purificationtechniques are not preferred in the present invention.

The availability of nucleic acids molecules encoding a protein enablesproduction of the protein using in vitro expression methods known in theart. For example, a cDNA or gene may be cloned into an appropriate invitro transcription vector, such a pSP64 or pSP65 for in vitrotranscription, followed by cell-free translation in a suitable cell-freetranslation system, such as wheat germ or rabbit reticulocytes. In vitrotranscription and translation systems are commercially available, e.g.,from Promega Biotech, Madison, Wis. or BRL, Rockville, Md.

Alternatively, according to a preferred embodiment, a selected peptideor protein may be produced by expression in a suitable procaryotic oreucaryotic system. For example, a DNA molecule, encoding a peptide orprotein component of the invention, or an entire composite targetedantigen of the invention, may be inserted into a plasmid vector adaptedfor expression in a bacterial cell, such as E. coli, or into abaculovirus vector for expression in an insect cell. Such vectorscomprise the regulatory elements necessary for expression of the DNA inthe host cell, positioned in such a manner as to permit expression ofthe DNA in the host cell. Such regulatory elements required forexpression include promoter sequences, transcription initiationsequences and, optionally, enhancer sequences.

A peptide or protein produced by gene expression in a recombinantprocaryotic or eucaryotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, so as to bereadily purified from the surrounding medium. If expression/secretionvectors are not used, an alternative approach involves purifying therecombinant protein by affinity separation, such as by immunologicalinteraction with antibodies that bind specifically to the recombinantprotein. Such methods are commonly used for isolating peptides andproteins.

D. Linking Separately-Made Proteins and/or Peptides

In an alternative embodiment, protein and/or peptide components of theinvention are synthesized separately, then conjugated using standardmethods known by those skilled in the art. For example, a syntheticpeptide may be chemically coupled to a protein usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBF). This reagentcross-links amino- and carboxy-terminal thiol groups in the peptide withlysine side chains present in the protein. Alternatively, a syntheticpeptide may be coupled to a protein using glutaraldehyde, a commoncross-linking agent. Another method for chemically coupling a peptide toa protein is through the use of carbodiimide and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDC). Asdescribed in greater detail in Example 2, this method was used toconjugate a C5a C-terminal decapeptide analog to serum amyloid A (SAA).Methods for joining two proteins together are also available.

The peptides or proteins of the invention, prepared by theaforementioned methods, may be analyzed according to standardprocedures. For example, they may be subjected to amino acid sequenceanalysis, mass spectra analysis or amino acid compositional analysisaccording to known methods.

E. General Formulae and Exemplary Compositions of the Invention

The APC-targeted antigens of the invention can comprise one or moreantigenic moieties, and likewise can comprise one or more targetingmoieties. Moreover, these moieties can be functionally linked in severalways. For instance, if “T” represents a targeting moiety, and “Ag”represents an antigenic moiety, the APC-targeted antigens of the presentinvention may be organized as follows:

-   -   Ag-T;    -   T-Ag;    -   T₁-Ag-T₂;    -   T₁-[Ag]_(n)-T₂ (wherein [Ag]_(n) represents a multiplicity of        antigens.

Examples of the general formulas set forth above include:

-   -   Ag-C5a agonist peptide;    -   IFNγ peptide-Ag;    -   IFNγ peptide [Ag]_(n)-C5a agonist peptide.    -   Other representative compositions of the invention include:    -   MUC1 Class I binding epitope—C5a agonist C-terminal peptide    -   Murine or human IFNγ peptide—MUC1 Class I binding epitope    -   Murine or human IFNγ peptide—MUC1 tandem repeat    -   MUC1 Class I epitope—C3dG peptide    -   SAA-K-Ahx —C5a C-terminal peptide (Ahx=εamino hexanoic acid).

It will be appreciated by persons skilled in the art that theAPC-targeted activating antigens of the invention may be adapted forinclusion of large or complex antigens. This may be accomplished, forexample, by inclusion of a “spacer” (such as the K-Ahx spacer moiety inthe exemplary compound above) between the antigen and the targetingmoiety. Such chemical modifications are familiar to biochemists.

II. Uses of APC-Targeted Activating Antigens

The APC-targeted activating antigens of the present invention have broadpotential for clinical applications in humans and animals. As discussedabove, a significant impediment to the development of vaccines and otherimmunotherapeutic agents is the apparent inability of particularantigens to be readily taken up and processed by antigen presentingcells. The compositions of the invention facilitate the specificdelivery of an antigen to a population of antigen presenting cells,whereupon the delivery mechanism (e.g., using as the targeting moiety areceptor ligand capable of transducing a biological signal)simultaneously activates the antigen presenting pathway. of the APC.Thus, the present invention enables development of vaccines and otherimmunotherapeutics that can specifically target any peptide antigen orother antigenic structure covalently attached to a ligand for a receptorpresent on an antigen presenting cell. It is believed that antigenslinked to ligands that selectivity bind to and activate a particularpopulation of APCs can not only generate an immune response, but caninfluence the nature of the immune response that is generated. Thus,immune responses that favor antibody, cellular, Th1 or Th2 responses,respectively, may be selectively generated. Vaccines may also bedeveloped with an array of such targeting moieties thereby serving totarget a selected antigen or antigens to several populations of APCs andsimultaneously activate these and other cells involved in various immunemodulatory pathways.

The ability to generate either antibody or cell mediated immuneresponses against different specific antigens has broad generalapplicability, and it is anticipated that the APC-targeted antigens ofthe invention will be extremely useful for these purposes. For example,antibody responses have been shown to be capable of protecting againstdifferent viral or bacterial infection, and antibodies are known toinactivate different toxins or toxic compounds that may affect the wellbeing of humans or animals. Different cell mediated immune responses canprovide protection against viral or other intracellular pathogens, andcan play a role in some anti-tumor responses. It is believed thatdifferent antigen presenting cells and the context in which these cellsare stimulated to present antigen (co-stimulation mediated by differentligand-receptor interactions) are important factors determining thenature of the above responses.

The targeted antigens of the present invention should find particularutility in the development of active specific immunotherapeutic agents(i.e., cancer “vaccines”) based on cancer-associated antigens. Forexample, it has been hypothesized that induction of strong cell-mediatedimmune responses (involving Th1 cells and/or cytotoxic T lymphocytes)would provide the most effective protection against various forms ofcancer. A vaccination strategy utilizing the APC-targeted antigens ofthe invention can be designed to induce this type of response. In thisregard, it is known that stimulation with some cytokines (IL-12, IFNγ)can induce predominantly Th1 type responses over Th2 type responses forcertain antigens.

As a step toward developing anti-cancer vaccines for clinical use, thecompositions of the invention can be used to advantage as research toolsto further explore the effect of stimulating a certain population ofAPCs with a tumor antigen and determining the effect on an anti-tumorimmune response. To this end, it should be noted that the presentapplication exemplifies targeted antigens comprising an epitope of aparticular tumor-specific antigen, Mucin-1.

Previous tumor vaccine formulations that aim to immunize patients withcompounds that are identical to compounds already produced by tumorshave proven to be of limited value, probably because tumors thatprogress have been selected for their lack of immunogenicity in theirrespective host (e.g., the host is tolerant to existing tumor antigens).Thus, one important challenge of producing effective tumor vaccines isgenerating reagents that counteract immunological tolerance totumor-associated antigens. One purpose of the APC-targeted antigensdescribed above is to induce in the immunized individual a responseagainst their tumor that is similar to that seen in individualsundergoing allograft rejection. In other words, the goal is to induce anautoimmune reaction against the tumor that is capable of destroying thetumor. The immunological parameters that regulate tolerance to tumorantigens are not well understood; nonetheless the compositions describedherein have the potential to counteract this tolerance and thus inducespecific immune responses that mediate tumor rejection.

The targeted antigens of the present invention will also find broadutility in the production of antibodies for use as immunodiagnostic andimmunotherapeutic agents. For immunodiagnostic purposes, antibodies arewidely used in various quantitative and qualitative assays for thedetection and measurement of biological molecules associated withdiseases or other pathological conditions. For reasons that often arenot well understood, it is sometimes difficult to generate antibodiesagainst certain biological molecules using conventional means. Thecompositions of the present invention provide an alternative means forinducing an animal to produce antibodies against a weakly-antigenic ornon-antigenic substances. The utility of the compositions of theinvention in this regard is shown clearly in Example 2, below, inconnection with serum amyloid A. The appearance and abundance of thisprotein in the body is strongly correlated with systemic inflammatorystress, which is a condition that is very difficult to quantitate. It isbelieved that quantitative assays for SAA levels would be an excellentindicator of general, systemic inflammation; therefore it would be ofbenefit to generate antibodies against the protein in a non-humanspecies. This protein has proved particularly recalcitrant to thegeneration of antibodies using conventional measures. As described inExample 2, a targeted antigen comprising SAA conjugated to a C5a peptideligand produced a significant antibody response in mice injected withthe conjugated molecule. In a similar fashion, targeted antigenscomprising any weakly-antigenic or non-antigenic component of interestcould be made and used to produce specific antibodies in laboratoryanimals, for use as immunodiagnostic reagents.

Antibodies for use as immunotherapeutic agents can also be generatedusing the compositions of the invention. As one example, there has beena great deal of recent interest in developing reagents capable ofdown-regulating or inhibiting the complement cascade to modulate localand systemic inflammatory responses. To this end, the C3a convertase,which is active early in the cascade, could provide an ideal target forcomplement inhibition. C3a convertase cleaves the peptide C3 into twocomponents, C3a and C3b, and therefore must be able to access thecleavage site on C3 in order to accomplish the result. Antibodiesdirected toward the C3a-C3b cleavage site are expected to be effectivein blocking access of C3a convertase to the cleavage site, therebyinhibiting this early step in the complement cascade. Such antibodiesmay be generated using a targeted antigen of the invention comprising,as the antigenic moiety, the short peptide sequence comprising theC3a/C3b cleavage site. The sequence could then be conjugated to anappropriate targeting moiety, such as the C5a C-terminal decapeptideagonists exemplified herein. Thus, the compositions would be-useful togenerate an immunotherapeutic agent (e.g., an antibody that blocks theactivity of C3a convertase) for treating an adverse inflammatorycondition.

The following examples are provided to describe the invention in furtherdetail. These examples are intended to illustrate the invention ingreater detail. They are not intended to limit the invention in any way.

EXAMPLE 1 Evaluation of Mucin Epitope (MUC1/C5a Agonist) Conjugate forRecruitment and Activation of Antigen Presenting Cells (APCs) andStimulation of an Immune Response in Mice

The C5a receptor is present on numerous antigen presenting cells,including monocytes, macrophages, dendritic cells, and other cell types.In this example, a composite peptide comprising a mucin epitope (MUCl)functionally linked to a decapeptide agonist analog of C5a correspondingto the C-terminal effector region of the natural factor was evaluatedfor its ability to activate the APCs thereby stimulating an immuneresponse in mice. This evaluation is based on the known property of C5areceptors to internalize and recycle in the antigen presenting cell,thereby acting as ideal candidates for delivering antigens to andsimultaneously activating signals in the APCs. Because C5a receptors areparticularly common on macrophages, monocytes and dendritic cells, it isbelieved that the use of a C5a agonist analog to bind C5a receptors willresult in preferential activation of these APCs.

i. Abbreviations. Except where noted, the single letter designation forthe amino acid residues is used: alanine is A; arginine is R; asparagineis N; aspartic acid is D; cystine is C; glutamine is Q; glutamic acid isE; glycine is G; histidine is H; isoleucine is I; leucine is L; lysineis K; methionine is M; phenylalanine is F; proline is P; serine is S;threonine is T; tryptophan is W; tyrosine is Y; and valine is V. Uppercase letters represent the L-amino acid isomer and lower case theD-isomer.

ii. Peptide synthesis, Purification and Characterization. The followingpeptides were synthesized according to standard solid-phasemethodologies on an Applied Biosystems (Foster City, Calif.) model 430 Apeptide synthesizer and characterized as previously described (7):

(1) The antigenic “juxta-membrane” (JM) epitope of the human mucin-1(MUC1), YKQGGFLGL (SEQ ID NO:6);

(2) The C5a C-terminal decapeptide agonist analog, YSFKPMPLaR (SEQ IDNO:1);

(3) The composite peptide YKQGGFLGLYSFKPMPLaR (SEQ ID NO:2), in whichthe JM epitope is positioned toward the amino terminus and the C5apeptide is positioned toward the carboxyl terminus; and

(4) The composite peptide YSFKPMPLaRKQGGFLGL (SEQ ID NO:5), in which theJM epitope of MUC1 is positioned toward the carboxyl terminus and theC5a analog is positioned toward the amino terminus.

Peptide 3 retains C5a biological activity, whereas peptide 4 does notbecause the biologically important carboxyl terminal end of the C5aanalog is blocked by the presence of the mucin epitope. As such, peptide4 serves as a control to determine the importance of the C5a biologicalactivity to the effectiveness of these peptides for immunizationpurposes.

Syntheses were performed on a 0.25 mmol scale on0-hydroxymethylphenoxymethyl polystyrene (HMP) resins (0.88 meq/gsubstitution). N^(α)-amino groups were protected with the base-labile9-fluorenylmethyloxycarbonyl (Fmoc) group. Side-chain functional groupswere protected as follows: Arg (Pmc or2,2,5,7,8-pentamethylchroman-6-sulfonyl); Asp (ot-butyl ester); Cys, Gin& His (Trt or trityl); Lys (Boc or t-butyloxycarbonyl); Ser & Tyr(t-butyl). Synthesis was initiated by the in situ coupling of theC-terminal residue (N^(α)-Fmoc-L-Arg(Pmc)) to the HMP resin in thepresence of excess N-N′-dicyclohexylcarbodiimide (DCC) and1-hydroxybenzotriazole (HOBT) with 4-dimethylaminopyridine (DMAP) as acoupling catalyst. Peptide chain elongation was accomplished byrepetitive Fmoc deprotection in 50% piperidine in NMP followed byresidue coupling in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU).

Side-chain deprotection and cleavage from the resin were achieved in asingle step acetolysis reaction by stirring the peptide-resin in asolution of 84% trifluoroacetic acid (TFA), 6% phenol, 2% ethanedithiol,4% thioanisole, and 4% water for 1.5 hr at room temperature. Freepeptide was precipitated from this solution by adding cold diethylether. The mixture was filtered through a scintered glass Buchner funnel(medium porosity) and the peptide/resin washed twice with cold ether toremove the thiol scavenger. The peptide was extracted by swirling thepeptide/resin in the funnel with 20-30 ml aliquots of 10% acetic acidfollowed by filtration. The extraction aliquots were combined, frozen,and lyophilized to yield the powdered form of the crude peptide.

Peptides were purified by preparative and analytical reverse-phase HPLCon columns packed with C₁₈-bonded silica. The details of this procedurehave been described by (4). All peptides were characterized by aminoacid compositional analysis and fast atom bombardment mass spectrometry(FAB-MS).

iii. Animal Models. The strains of mice used for this example wereinbred females 6 to 12 week old C57B16(H-2^(b)) and Balb/c (H-2^(d)),which were obtained from Jackson labs (Bar Harbor, Me.). These twostrains which differ in H-2 haplotypes, were used in this example todemonstrate that the observed antibody responses were not a result ofthe selection or creation of an unique immunogenic epitopecharacteristic of the sequence of the proteins of the MHC class I andclass II molecules important for antigen processing in one mouse strainor another. The MUC1 peptide selected for these studies contained amotif that may bind to the H-2K^(b) molecule of the C57B16 mice;therefore, a strain of mouse that lacked this class I molecule bindingmotif (Balb/c) was also studied to determine the relative contributionof the class I binding motif to the antigen presentation properties ofthese peptides.

iv. Immunization protocol. Preimmune sera were obtained from mice, whichwere subsequently immunized intraperitoneally with 100 μg of theindicated peptide with RIBI adjuvant (MPL+TDM+CWS) (SigmaImmunochemicals). Animals were boosted twice at two week intervals usingthe same injection procedure. Sera were obtained following threeimmunizations (at 6 weeks).

v. Analysis of serum antibody responses. For radioimmunoassay (RIA),anti-peptide antibodies were determined, before and at different timepoints after immunization, in 96-well microtiter plates (DynatechLaboratories, Inc.). Plates were coated with 50 μl of a 100 μg/mlappropriate peptide in phosphate-buffered saline (PBS) pH 7.4 solutionovernight at 4° C. The wells were blocked by incubation with 5% dry milkin PBS pH 7.4 for at least two hours. Anti-peptide antibody titers weredetermined using serial dilutions of sera. The sera were diluted withPBS containing 0.05% Tween-20, pH 7.4 (washing buffer) and 50 μl of eachdilution was incubated at 37° C. for 1 hour. The wells were thendrained, washed 4 times with PBS-Tween and 50 μl of ¹²⁵I-goat anti-mouseAb (1−2×10⁴ cpm/well) was added and incubated for 1 hr at 25° C. Afterwashing, specific radioactivity was recorded in a gamma counter (1272CliniGamma, LKB).

Anti-peptide antibody isotype titers were determined by enzyme-linkedimmunosorbent assay (ELISA) carried out in 96-well microtiter plates.The plates were coated with 100 Ag/ml of appropriate peptide in PBS, pH7.4, and incubated overnight. The wells were blocked with 5% dry milk inPBS pH 7.4 for at least two hours. Anti-peptide titers were determinedusing serial dilutions of sera as described above. After the plates werewashed 4 times, 50 μl of a 1:100 dilution of rabbit anti-mouse IgA,IgG1, IgG2a, IgG2b, IgG3 and IgM (Zymed) was added to each well andincubated at 25° C. for 1 hour. The plates were washed 4 times withwashing buffer and 50 μl of 1:500 goat anti-rabbit conjugated toperoxidase (Zymed) was incubated at 37° C. for 1 hour. Again, the plateswere washed 4 times with washing buffer and bound enzyme was detected bythe addition of 50 μl 1 mg/ml p-nitrophenyl phosphate (Sigma) in 10%diethanolamine (Sigma) pH 9.4. The reaction was stopped by the additionof 50 μl of 0.5 M sodium hydroxide and absorbance values (A₄₀₅) weredetermined on Titertek Multiskan (Flow Laboratories, Irvine, Scotland).

vi. Experimental groups. Experimental groups were as follows:

-   -   Group A. mice immunized with peptide (1)    -   Group B. mice immunized with peptide (2)    -   Group C. mice immunized with peptide (1) plus peptide (2)    -   Group D. mice immunized with peptide (3)    -   Group E. mice immunized with peptide (4).

The results of the experimental protocols are set forth in FIGS. 1 and2. As can be seen in the Figures, the mice in Groups A, B, C and Eproduced no appreciable increase in antibody response to inoculationwith MUC1 epitope (Group A), C5a agonist peptide (Group B), MUC1 epitopecombined with, but not conjugated to, C5a agonist peptide (Group C), orMUC1 epitope conjugated to the C5a agonist peptide at its C-terminus,rather than its N-terminus (thereby blocking C5a biological activity)(Group E). Only mice inoculated with the MUC1 epitope/C5a agonistpeptide conjugate of the present invention (Group D) generated anappreciable antibody response. Furthermore, this stimulation wassignificant. It is clear from these results that inoculation with theconjugated MUC1 epitope/C5a agonist peptide was far more efficient instimulating a general immune response (i.e., production of antibodies)than was inoculation with either peptide alone, or both peptidestogether, but not conjugated, or peptides conjugated in the oppositeorientation.

There are several significant conclusions that can be drawn based onthese results. The fact that both Balb/c and C57B16 mice showed antibodyresponses to peptide 3 suggests that the antigen presenting effect isnot restricted by MHC haplotype. The fact that immune responses were notproduced to peptide 4, or to mixtures of peptide 1 and 2, but thatsubstantial responses were produced to peptide 3, suggest that theeffect is mediated by the C5a moiety of the peptide and that the immuneresponse results from the simultaneous delivery of antigen peptide andC5a mediated activation sianals to antigen presenting cells.

The isotypes of the anti-peptide antibodies produced in the immunizedmice were determined (FIG. 3) and were found to consist of IgM, IgG2a,and IgG2b. This suggests that the immunogenic peptide is producing Tcell-dependent responses, which generally require antigen processing andpresentation. Data presented in FIG. 4 show that the antibody responseto peptide 3 includes a high percentage of antibodies that are-specificfor the MUC1 epitope that was the antigen moiety of these studies.

EXAMPLE 2 Evaluation of-Serum Amyloid A/C5a Peptide Conjugates forRecruitment and Activation of APCs and Stimulation of Immune Response inRats

Serum amyloid A is an acute-phase stress response protein generated bythe liver. Along with other acute phase proteins, SAA is secreted inresponse to systemic inflammatory stress as a protective measure. SAA isof interest because it appears to be an excellent indicator of general,systemic inflammation, which is a phenomenon that is very difficult toquantitate. Because serum levels of SAA have been observed to parallelthe rise and fall of the systemic inflammatory response, quantitation ofserum levels of this peptide would provide an effective means ofassessing inflammation. One way to accomplish this is to developantibodies against SAA that could be used for quantitation such as in anELISA assay. However, SAA has been particularly recalcitrant to thegeneration of antibodies against it. In this example, an evaluation wasmade of the ability of SAA conjugated to a C5a C-terminal analog (asdescribed in Example 1) to activate antigen producing cells and producean antibody response in rats.

i. Production and preparation of proteins and peptides. The C-terminalC5a analog K-Ahx-YSFKPMPLaR (SEQ ID NO:8) (AhX is aminohexanoic acid,which is a linear aliphatic spacer moiety) was produced as described inExample 1. The aliphatic spacer moiety was included to separate thecritical receptor-binding C5a analog from the bulky protein to beattached to the amino terminus.

Serum amyloid A was conjugated to the C5a peptide analogs according tothe following method. SAA (100 μg) was reacted with a 50-fold molarexcess of a water soluble carbodiimide,1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodiide (EDC), in 200μl of phosphate buffered saline, pH 7.5, at room temperature for 30minutes. A 50-fold molar excess of the peptide (K-Ahx-YSFKPMPLaR) (SEQID NO:8) and a 100-fold molar excess of a base diisopropylethyl amine(DIEA) were added to this solution. Water was added to the solution tobring the reaction mixture to a volume of 400 μl. This solution wasstirred overnight at room temperature and then lyophilized to a drypowder. The powder was diluted to the appropriate volume with water togenerate the stock mixture used for inoculating the animals.

ii. Experimental protocols. Rats were injected intraperitoneally with aninoculant comprising the SAA/C5a peptide conjugates inphosphate-buffered saline with or without RIBI adjuvant. Boosterinjections were given two and five weeks after the initial injections.The rats were sacrificed seven weeks after the initial injection andanti-mucin antibody production was assessed from the serum titers, asdescribed in Example 1.

Significant anti-SAA antibody was produced from both groups of rats,whether or not RIBI adjuvant was included in the inoculation. Asvisualized by gel electrophoresis and autoradiography of anti-SAAantibody eluted from the plate assays, it appeared that anti-SAAantibody titers were essentially equivalent, or slightly higher, in ratsinoculated with SAA/C5a peptide conjugate in the absence of RIBIadjuvant as compared to the same inoculation without the adjuvant. Thus,antigenic conjugates comprising the C5a peptide analog are useful forgenerating antibodies against large proteins, as well as against smallerpeptide fragments, such as those described in Example 1. Moreover, thesuccessful generation of anti-SAA antibodies utilizing this method isparticularly promising for purposes of producing antibodies againstweakly- or non-antigenic peptides or proteins.

EXAMPLE 3 Production and Characterization of Site-Directed NeutralizingAntibodies Specific for a Peptide κR(33-52) from the PredictedAmino-Terminal Region of the Human Kappa Receptor

Receptors for human opioid peptide hormones have been described onnumerous cell types. The receptors for μ, κ, and δ ligands have recentlybeen cloned from genomic and cDNA libraries derived from normal tissueand cell lines. Considerable homology exists among the μ, κ, and δreceptors, except for the N-terminal regions of the receptors. The Nterminal region of the human kappa receptor (amino acid residues 1-100)is relatively hydrophilic and would be predicted to be exposed on thesurface of the cell membrane. A 20 residue peptide [κR(33-52)], waschosen and used to raise a site directed peptide specific polyclonalantibody (5).

The method of production of a polyclonal antiserum in rabbits using themolecular adjuvant, C5a-agonist peptide conjugated to the κR epitope isset forth below. The binding specificity and biological activities ofthe resulting polycolonal antiserum raised to the predictedextracellular region of the human kappa receptor (κR) are also describedbelow.

i. Construction of Targeted-Immunogen. A peptide construct consisting ofthe κR(33-52) (FPGWAEPDSNGSAGSEDAQL) (SEQ ID NO:9) covalently attachedto the N-terminal end of a conformationally biased, C5a complementfragment agonist analogue peptide (YSFKPMPLaR) (SEQ ID NO:1) wassynthesized according to the methods in Example 1 and as previouslyreported (7).

ii. Preparation of anti-κR(33-52) Antiserum and Peptide-Specific ELIBA.Rabbits were immunized s.c. with 500 μg of FPGWAEPDSNGSAGSEDAQLYSFKPMLaRconstruct (SEQ ID NO:10) in complete Freund's adjuvant (GIBCO, GrandIsland, N.Y.) on day O followed by booster injections on days 30 and 60in incomplete Freund's adjuvant. Serum was collected starting 75 daysafter the initial immunization.

The presence of anti-peptide antibody was determined by using a peptidespecific ELISA utilizing the free κR(33-52) peptide as previouslydescribed (8). Anti-κR(33-52) and normal rabbit γ-globulin (RGG) werepurified by protein A Sepharose chromatography (Sigma) (8) prior to use.

iii. Cells and culture conditions. The neuroblastoma cell SK-N-SH (HTB11), ductal breast cell carcinoma T47D (HTB 133), Jurkat T cellleukemia, (TIB 152), U937 histolytic lymphoma (CRL1593), THP 1 humanmonocyte (TIB 202), EBV-transformed B cells SKW 6.4 (TIB 215) and CESS(TIB 190) (American Type Culture Collection, Rockville, Md.) werecultured in DMEM or RPMI 1640 supplemented with 10% fetal calf serum, 25mM HEPES, 1 mM L-glutamine, 2 mM Na pyruvate, 50 U penicillin and 50μg/ml streptomycin. The human neuronal precursor cells NT2 (Stratagene,La Jolla, Calif.) were cultured in Opti-MEM (Gibco) supplemented asabove. All cultures were incubated at 37° C. in a humidified chamberwith 7.5% CO₂.

Peripheral blood derived mononuclear cells were obtained from healthymale and female volunteers, isolated by Ficoll-Hypaque(tm) densitygradient centrifugation and enriched for macrophage by adherence toplastic.

iv. Flow Cytometry. Single-color flow cytometry analysis of cells(1×10⁶) in PBS containing 1% bovine calf serum and 0.1% sodium azide(staining buffer) were preincubated 30 minutes at 4° C. in the presenceof 20% normal human serum. The cells were washed and incubated withanti-κR(33-52) or RGG for 30 minutes at 4° C., washed and labeled withPI-conjugated donkey (Fab′)2 fragments of antirabbit IgG (Zymed, S. SanFrancisco, Calif.) for 30 minutes at 4° C. (8). For dual color analysisFITC-conjugated anti-CD3 or anti-CD14 (Pharmingen, San Diego, Calif.)were also included in the second step. Cells (1×10⁴) were analyzed on aFACScan (Becton Dickinson, Mountain View, Calif.) and data were analyzedwith the Cell Quest software as previously described (8).

V. Measurement of cell proliferation. Peripheral blood mononuclear cell(PBMC) were pulsed on day 2 of culture with ³H-thymidine and 18 hourslater the cells harvested on glass fiber filters and processed forscintillation counting. Experiments were performed three times and eachsample done in triplicate.

vi. Measurement of IgG Secretion. Relative IgG levels in culturesupernatants were determined by indirect ELISA as previously described(9). Supernatants from PBMC cultures were collected after 10 days andassayed for the presence of IgG. Numbers represent the mean CPM+/−SDfrom triplicate samples. Experiments were performed at least threetimes.

vii. Characterization of Anti-κR Peptide Antiserum. Serum from rabbitsimmunized with the κR(33-52)YSFPMPLaR construct (SEQ ID NO:10) andnormal rabbit serum were assayed for the ability to recognize platebound κR(33-52) (SEQ ID NO:9) in ELISA. The results show that serum fromrabbits immunized with the κR(33-52)YSFPMPLaR construct (SEQ ID NO:10)bound free κR(33-52) peptide (SEQ ID NO:9) in a dose dependent fashion.The titer was approximately 10⁵. In contrast, serum from unimmunizedrabbits failed to bind this peptide. Serum samples from immunized andunimmunized rabbits were subjected to protein A-Sepharose chromatographyand the column eluates were assessed for κR(33-52) (SEQ ID NO:9)specific antibody. The results indicate that protein A-purified antibodyderived from rabbits immunized with the κR(33-52)YSFPMPLaR construct(SEQ ID NO:10) binding to free κR(33-52) (SEQ ID NO:9) was detectable atantibody concentrations less than 0.1 ng/ml. In contrast, RGG failed tobind the free peptide. The results from multiple bleedings indicatedthat the ED₅₀ titer ranged between 1-10 ng/ml. These results indicatethat rabbits immunized with κR(33-52)YSFPMPLaR (SEQ ID NO:10) containedhigh titer, κR(33-52) peptide specific antibody.

viii. Binding of anti-R (33-52) antibody to cells expressing human κR.To determine whether the polyclonal anti-κR(33-52) antibodies bound tocells expressing the κR, a variety of mononuclear cell lines and normalhuman mononuclear cells were first assayed for the presence of the κreceptor specific mRNA by RT-PCR. RNA samples isolated from neuronalcell lines NT2, U937, Jurkat, T47D, normal human PBMC, and enrichedhuman macrophage were subjected to RT-PCR analysis with 5′ sense and 3′antisense primers specific for the 3′ region of the cloned κR andB-actin. All of the cell lines or cell fractions, except for the T47Dcell line, were positive for the κ-receptor specific PCR product, asexpected based on the primer sequences used (5).

Experiments were performed to determine whether anti-κR(33-52) bound tocells expressing κR specific mRNA. The results of single color flowcytometric analysis for several cell lines are shown in Table 2. Flowcytometric measurements were conducted with human cell linesrepresentative of macrophage (U937), T lymphocytes (Jurkat), and Blymphocytes (SKW 6.4 and CESS). The results indicate that anti-κR(33-52)bound all three cell types. Anti-κR(33-52) bound to U937 cells to thegreatest extent (MFI=231) compared to normal RGG (MFI=38). As usedherein MFI refers to mean fluorescence intensity. Comparison ofanti-κR(33-52) and RGG binding to the Jurkat line indicatedapproximately a 3-fold shift in MFI (MFI-18 vs. MFI-6). Similar resultswere obtained with the two B lymphocyte-like cell lines (SKW 6.4 andCESS). comparison of anti-κR(33-52) and RGG binding to the SKW 6.4 lineindicated approximately a 3-fold shift in MFI (MFI=19 vs. MFI=6). Theneuronal cell line was also specifically bound by the anti-κR(33-52) asindicated by a 3-fold shift in the MFI over the RGG. Finally, based onthe lack of expression of κR-specific mRNA from the human breastcarcinoma cell line (T47D), this cell line was assessed for its abilityto bind to anti-κR(33-52) by flow cytometric analysis. The lack of a κRexpression on T47D cells was confirmed by the fact that anti-κR(33-52)and RGG bound to these cells in an almost identical fashion. As apositive control, anti-κR(33-52) and RGG were assessed for their abilityto bind to an additional human macrophage-like cell line (THP 1).Comparison of anti-κR(33-52) and RGG binding to this cell line resultedin a significant shift in MFI (MFI=190 vs. MFI=8). These results confirmthe specificity of anti-κR(33-52) for the human κR.

TABLE 1 Selected cell type binding of anti-κR(33-52) antibodies producedin rabbits immunized with C5a-agonist peptide conjugated to theκR(33-52) sequence as assessed by single channel color flow cytometricanalysis. Mean Channel Intensity Cell Line Cell Type RGG anti-κR Ab NT2Neuronal 9 19 U937 Macrophage 38 231 Jurkat T-lymphocyte 6 18 SKW 6.4B-lymphocyte 6 19 CESS ″ <10 >10 Controls T47D (negative) Human ~3 ~3Breast Carcinoma THP1 (positive) Macrophage 8 190

Analysis of intact human PBMC indicated that these cells express mRNAfor a “κ-like” R (5). Dual color flow cytometric analysis was utilizedto assay for the binding of anti-κR(33-52) to normal human macrophage(CD14+) and T lymphocytes (CD3+). It was observed that both macrophageand T lymphocytes bound anti-κR(33-52) antibody. Anti-κR(33-52) and RGGwere assessed for binding to CD14+ PBMC. The results indicate thatanti-κR(33-52) bound CD14+ cells with a 15-fold increase compared tonormal RGG (MFI=320 vs. MFI=21). Anti-κR(33-52) was also found to bindCD3+ cells (MFI=19 vs. RGG MFI=3) albeit less than CD14+ cells. Theseresults indicate that anti-κR(33-52) binds normal PBMC-derivedmononuclear cells as well as mononuclear cell lines, which expressκR-specific mRNA.

ix. Neutralization of U50,488H-mediated suppression of lymphocyteproliferation by anti-κR(32-52) antibody in vitro. The results ofpublished studies have shown that opioid peptide-induced regulation ofin vitro immune responses can occur via specific receptor-ligandinteractions. More specifically, it has been shown that the κR-selectiveagonist U50,488H is capable of suppressing SAC-induced lymphocyteproliferation by human PBMC cultures (6). The inhibition of lymphocyteactivation by U50,488H has also been shown to be reversed by theκR-selective antagonist nor-BNI. To determine whether anti-κR(33-52) wascapable of acting as an κR selective antagonist and neutralizingU50,488H-mediated suppression, PBMC cultures were preincubated withvarious concentrations of protein A purified anti-κR(32-52) prior theaddition of SAC and US50,488H. U50,488H suppresses SAC-inducedlymphocyte proliferation in a dose dependent fashion (5). Maximalsuppression was obtained when U50,488H was used at a concentration of10⁻⁶ M. PBMC cultures were preincubated with various concentrations ofanti-κR(33-52) (1-50 μg/ml), followed by the addition of U50,488H plusSAC, and proliferation measured on day 3 of culture. Anti-κR(33-55) wasfound to neutralize U50,488H-mediated suppression of SAC-inducedlymphocyte proliferation in a dose dependent fashion. In contrast,identical concentrations of normal RGG failed to inhibit κR selectiveagonist mediated immunosuppression.

Since SAC has been shown to induce both T and B lymphocyteproliferation, similar experiments were conducted with the T cellmitogen PHA. Anti-κR(33-52) was also able to neutralize the ability ofU50,488H to suppress mitogen-induced T cell proliferation. U50,488H(10⁻⁶ M) suppressed PHA-induced T cell proliferation by 85%. Thissuppression was reversed by preincubating the cells with anti-κR(33-52).Preincubation of PBMC with normal RGG failed to block U50,488H-mediatedsuppression of T cell proliferation.

Anti-κR(33-52) does not appear to directly modulate lymphocyteproliferation. The co-culture of PBMC with anti-κR(33-52), in theabsence of mitogen, failed to stimulate the cells above the mediacontrol. Moreover, the combination of anti-κR(33-52) and PHA or SAC didnot result in increased cell proliferation compared to PBMC culturesreceiving mitogen only.

x. Neutralization of U50,488N-mediated suppression of IgG synthesis byanti-κR(32-52) antibody in vitro. In addition to lymphocyteproliferation, U50,488H is a potent inhibitor of SAC-induced IgGsynthesis in human PBMC cultures (6). To determine whetheranti-κR(32-52) was capable of neutralizing the suppression of IgGsynthesis, PBMC were preincubated with anti-κR(32-52) followed by theaddition of U50,488H and SAC, and IgG levels measured on day 10. Resultsindicate that U50,488H at 10⁻⁸ M and 10−7 M inhibited IgG synthesis by67% and 85% respectively (5). The inclusion of anti-κR(32-52) in culturewas found to neutralize suppression of SAC induced IgG synthesis in adose dependent manner. In contrast, similar concentrations of normal RGGfailed to neutralize the observed suppression.

To assess the specificity of anti-κR(32-52) antibody, PBMC wereincubated with specific antibody or RGG followed by co-culture withU50,488H or the μ receptor selective agonist (DAGO) and IgG productionmeasured by ELISA. The results indicate that, whereas, anti-κR(32-52)neutralized U50,488H-mediated inhibition of SAC-induced IgG synthesis,anti-κR(32-52) was unable to neutralize DAGO-mediated suppression of IgGsynthesis.

These results indicate that in addition to binding lymphocytes andmacrophage, anti-κR(32-52) is capable of neutralizing the ability of aκR selective agonist (U50,488H), but not a AR selective agonist (DAGO).Additionally the antibody demonstrated significant inhibition of bothlymphocyte proliferation and differentiation to antibody synthesis.These results further demonstrate the specificity of anti-κR(33-52) forthe human kappa receptor.

As can be seen from the antibody binding data presented above, the sitedirected polycolonal antibodies raised in rabbits using the C5a-agonistform of the molecular adjuvant conjugated to the κ receptor sequencewere capable of binding to normal human cells and cell lines expressingmRNA specific for the human κ receptor. Flow cytometric analysis of aneuronal cell line (NT2), normal blood-derived CD14+ monocytes,monocyte-like cell lines (U937 and THP1), normal blood derived CD3+ Tcells and a T cell line (Jurkat), and human B cell lines (SKW6.4 andCESS) revealed that the cells were all bound by anti-κR(33-52) in aspecific manner. The anti-κR(33-52) did not bind to a cell linedetermined not to express mRNA for the human κ receptor.

Anti-κR(32-52) was found to specifically neutralize κR-selective agonist(U50,488H)-mediated inhibition of lymphocyte activation. The antiserumwas found to neutralize, in a dose dependent manner, U50,488H-mediatedinhibition of: 1) SAC-induced lymphocyte proliferation; 2) PHA-inducedlymphocyte proliferation and; 3) SAC-induced IgG synthesis. In contrast,DAGO-mediated suppression of SAC-induced IgG production was not affectedby anti-κR(32-52). These results suggest that this site directedpolyclonal antiserum specifically interacts with the human κR on PBMC.The results presented indicate that polyclonal anti-κR(32-52) antibodiesinteract with the exposed N-terminal region of the κR. While thisantiserum effectively blocked U50,488H-mediated lymphocyte activation,it did not activate macrophage or lymphocytes.

While anti-κ opioid receptor antibodies are exemplified above,conjugation of C5a agonist peptide to peptides corresponding to μ and Δspecific peptides has resulted in the successful generation of specificantibodies to the μ and Δ epitopes.

EXAMPLE 4 Comparison of Immunogenicity of Epitope-C5a Agonist Constructswith Epitope-KLH Conjugates.

The following experiment was performed in order to compare the potencyof the molecular adjuvant of the present invention with a widely usedmethod for enhancing the immune response to peptide epitopes. Theobjective was a direct comparison of the response to a construct of MUC1epitope-C5a agonist and the same epitope conjugated to keyhole limpethemocyanin (KLH) in mice. The results are summarized in Table 2.

TABLE 2 MUC1 Specific Ab Isotype Titers Produced with DifferentImmunogens. Ab Isotypes and Titers^(a) IgA IgG1 IgG2a IgG2b IgG3 IgMYKQGGFLGLYSFKPMPLaR^(b) (SEQ ID NO:2) 0 0 1260 1780 0 6310 (5/5) (5/5)(5/5) YKQGGFLGL-KLH^(c) (SEQ ID NO:6-KLH) 0 100 0 0 0 5010 (2/5) (4/5)^(a)Sera were screened against MUC1 peptide and mean titer values ofresponders are shown. Parentheses indicate the number of responders. Abtiter is defined as the sera dilution within the linear range at whichspecific reactivity is lost. ^(b)Five C57BL6 mice were immunized andboosted with YKQGGFLGLYSFKPMPLaR (SEQ ID NO:2) and sera were obtained asindicated in the Material and Methods section. Standard error ofresponder titer values was less than 32% for all isotypes. ^(c)FiveC57BL6 mice were immunized and boosted with YKQGGFLGL-KLH (SEQ ID NO:6conjugated to KLH) and sera were obtained as indicated in the Materialsand Methods section. Standard error of responder titer values was lessthen 25% for IgM and less than 40% for IgG1.

A similar experiment was performed in rabbits. The immunogens used inrabbits were the κ- and μ-opioid receptor epitopes, FPGWAEPDSNGSAGSEDAQL(SEQ ID NO:9) and GDLSDPCGNRTNLGGRDSL (SEQ ID No:11), respectively. Theserum antibody titer and antibody subtypes produced in rabbits injectedwith the two compositions containing the different immunogens werecompared.

i. Peptide conjugates. In one instance the epitopes were conjugated toKLH via a lysine residue added synthetically to the N-terminus of theepitope along with an alanine residue which acted as a spacer. In thisexperiment, glutaraldehyde was used to effect conjugation. In theanother case,.the epitopes were linked to the N-terminal end of the C5aagonist YSFKPMPLaR (SEQ ID NO:1) using the solid phase peptide syntheticmethodologies described above in example 1.

ii. Immunization protocol for rabbits. Rabbits were immunized s.c. with500 μg of either the epitope-KLH or the epitope-YSFKPMPLaR (epitope-SEQID NO:1) constructs in compete Freund's adjuvant (GIBCO, Grand Island,N.Y.). Booster injections were administered on days 30 and 60 inincomplete Freund's adjuvant. Serum was collected starting at day 60post-immunization.

iii. Antibody determination. The presence of rabbit IgG specific for thepeptide epitopes was determined by ELISA as previously described (8).

Rabbits immunized with the epitope-C5a agonist generated high titer IgGAbs specific for the opioid receptor peptide epitopes. The rabbitsimmunized with the opioid receptor epitopes conjugated to the carrierprotein KLH also produced high titer antibodies specific epitopes towhich they were injected. These results demonstrate that the decapeptideC5a-agonist was as effective as the large molecular weight protein, KLH,conjugated to the epitope at inducing specific anti-peptide antibodiesin non-rodent species.

REFERENCES

-   1. Rammensee et al. (1993) “Peptides Naturally Presented by MHC    Class I Molecules,” Ann. Rev. Imm. 11:213-244.-   2. Ausubel et al., “Current Protocols in Molecular Biology,” John    Wiley & Sons, Inc., 1995.-   3. Barclay, et al., (1993) The Leucocyte Antigen Facts Book.    Academic Press, Harcourt Brace and Co., London.-   4. Ember, J. A., Sanderson, S. D., Taylor, S. M., Sawahara, M., and    Hugli, T. I. (1992) “Biological activity of synthetic analogues of    C5a anaphylatoxin”. J. Immunol. 148: 3165-3173.-   5. Robert R Buchner, Shawn M. Vogen, Wolfgang Fischer., Marilyn L.    Thoman, Sam D. Sanderson, and Edward L. Morgan. (1996) “Anti-Human    kappa opioid receptor antibodies characterization of site-directed    neutralizing antibodies specific for a peptide κR(33-52) derived    from the predicted amino-terminal region of the human kappa    receptor”, J. Immunol. (In press).-   6. Morgan, E. L. (1996) “Regulation of human B lymphocyte activation    by opioid peptide hormones. Inhibition of IgG production by opioid    receptor class (μ-, κ-, and , δ-) selective agonists”, J.    Neuroimmunol. 65:21.-   7. Sanderson, S. D., L Kirnarsky, S. A. Sherman, J. A. Ember, A. M.    Finch, and S. M. Taylor. (1994) “Decapeptide agonists of human C5a:    the relationship between conformation and spasmogenic and platelet    aggregatory responses”, J. Med. Chem. 38: 3171-3180.-   8. Morgan, E. L., J. A. Ember, S. D. Sanderson, W. Scholz, R.    Buchner, RD. Ye, T. E. Hugli. (1993) “Anti-C5a receptor    antibodies. I. Characterization of neutralizing antibodies specific    for the human C5a receptor”. J. Immunol. 151: 377.-   9. Hobbs, M. V., R. A. Houghten, J. A. Janda, W. O. Weigle,    and E. L. Morgan, E. L. (1989) “Induction of human B cell    differentiation by Fc region activators. I. Identification of an    active tetrapeptide”, Clinical Immunol. Immunopathol. 50:251.

While certain preferred embodiments of the present invention have beendescribed and specifically exemplified above, it is not intended thatthe invention be limited to such embodiments. Various modifications maybe made to the invention without departing from the scope and spiritthereof as set forth in the following claims.

1. A method for the production of antibodies to an immunogen,comprising: a) immunizing an animal with an immunogenically effectiveamount of a molecular adjuvant, said molecular adjuvant comprising atargeting ligand having binding affinity for a characteristicdeterminant of an antigen presenting cell, said targeting ligand beingcovalently linked to an immunogen, whereby binding of said molecularadjuvant to said antigen presenting cell determinant activates saidantigen presenting cell, effecting delivery of said immunogen to anantigen presenting pathway of said antigen presenting cell, wherein saidcharacteristic determinant is a C5a receptor, and wherein said targetingligand is at the C-terminus of said molecular adjuvant; b) isolatingantibodies from sera of said animal; and c) recovering said isolatedantibodies.
 2. The method of claim 1, wherein said targeting ligandbinds specifically to a C5a receptor and is selected from the groupconsisting of C5a, the C-terminal ten residues of C5a, and a peptideagonist analog of the C-terminal ten residues of C5a.
 3. The method ofclaim 2, wherein said targeting ligand is a peptide comprising thesequence of SEQ ID NO:
 1. 4. The method of claim 1, wherein saidtargeting ligand and said immunogen are linked by a spacer moiety. 5.The method of claim 1, wherein said immunogen comprises at least onesubstance selected from the group consisting of peptide, glycopeptide,phosphopeptide, lipopeptide protein, glycoprotein, phosphoprotein,lipoprotein carbohydrate, nucleic acid, and lipid.
 6. The method ofclaim 5, wherein said immunogen comprises a peptide.
 7. The method ofclaim 6, wherein said peptide comprises an epitope of human mucin-1. 8.The method of claim 5, wherein said immunogen comprises a protein.